论文部分内容阅读
研究了基于荧光定量PCR方法的肝素粗品种属来源质控方法,用于肝素药品生产企业的原料验收质量控制。该方法采用实时定量PCR法,针对猪、牛、绵羊、山羊基因组特有的基因序列设计引物与探针,采用聚合酶链式反应对目标基因进行扩增,利用扩增曲线的Ct值绘制标准曲线,通过未知样品的Ct值对未知样品初始模板中的基因进行定量。测定时通过肝素酶处理肝素原料,以消除肝素介导的PCR抑制效应。结果:该方法在本实验室条件下具有较好的专属性、准确度、精密度和耐用性:猪基因在30~0.06 ng/μL范围内呈良好线性,牛、绵羊、山羊基因在0.6~0.000 3 ng/μL范围内呈良好线性;猪基因的定量限为0.001 ng/μL,牛、绵羊、山羊基因的定量限为0.000 3 ng/μL。本质控方法较基因组纯化试剂盒提纯方法具有保证被检基因的真实性、原始性、完整性等优点。
The quality control of raw materials for heparin production based on fluorescence quantitative PCR was studied. The method uses real-time quantitative PCR method to design primers and probes for the gene sequences unique to pigs, cattle, sheep and goat genomes, amplifies the target gene by polymerase chain reaction, and draws a standard curve by using the Ct value of the amplification curve , The unknown template initial template of the gene was quantified by the Ct value of the unknown sample. The heparin material was assayed by heparinase to eliminate heparin-mediated PCR inhibition. Results: The method has good specificity, accuracy, precision and durability under the laboratory conditions. The results showed that the porcine gene showed a good linearity in the range of 30 ~ 0.06 ng / μL, while that of cattle, sheep and goat was in the range of 0.6 ~ 0.000 3 ng / μL. The limit of quantification for porcine was 0.001 ng / μL. The limit of quantification for bovine, sheep and goat was 0.000 3 ng / μL. The quality control method than the genome purification kit purification method to ensure that the seizure of the authenticity of the gene, the original, completeness and so on.